Large quantities of genomic and cDNA sequences have been determined with respect to a number of organisms by gene sequencing projects. In a plant model, Arabidopsis thaliana, the complete genomic sequences of two chromosomes have been determined (Lin, X. et al., (1999), Nature 402, 761-768; and Mayer, K. et al., (1999), Nature 402, 769-777).
The expressed sequence tag (EST) project also has greatly contributed to the discovery of expression genes (Hofte, H. et al., (1993), Plant J. 4, 1051-1061; Newman, T. et al., (1994), Plant Physiol. 106, 1241-1255; and Cooke, R. et al., (1996), Plant J. 9, 101-124; and Asamizu, E. et al., (2000), DNA Res. 7, 175-180). For example, the database of EST (dbEST) of the National Center for Biotechnology Information(NCBI) includes partial cDNA sequences, in which more than half (about 28,000 genes) of the total genes are reproduced, (as estimated from the gene content of Arabidopsis thaliana chromosome 2 completely sequenced [Lin. X. et al., (1999), Nature 402, 761-768]).
Recently, microarray (DNA chip) technology has become a useful tool for analyzing genome-scale gene expression (Schena, M. et al., (1995), Science 270, 467-470; Eisen M. B. and Brown, P. O. (1999), Methods Enzymol. 303, 179-205). In the technology using a DNA chip, cDNA sequences are arrayed on a slide glass in a density of not smaller than 1,000 genes/cm2. The cDNA sequences thus arrayed are hybridized simultaneously with a pair of cDNA probes tagged with two color fluorescent labels, which have been prepared from RNA samples of different types of cells or tissues. In this manner, a large amount of genes can be directly analyzed and compared for gene expression. This technology was demonstrated for the first time by analyzing 48 Arabidopsis genes for differential expression in root and shoots (Schene, M. et al., (1995), Science 270, 467-470). Furthermore, a microarray was used in investigating 1,000 clones randomly taken from a human cDNA library in order to identify a novel gene responsive to heat shock and protein kinase C activation (Schena, M. et al., (1996), Proc. Natl. Acad. Sci. USA, 93, 10614-10619).
In another method, a DNA chip is used in analyzing the expression profile of an inflammatory-disease associated gene under various induction conditions (Heller, R. A. et al., (1997), Proc. Natl. Acad. Sci. USA, 94, 2150-2155). Furthermore, using a microarray, a yeast genome having more than 6,000 coding sequences has been analyzed for dynamic expression (DeRisi, J. L. et al., (1997) Science, 278, 680-686; and Wodicka, L. et al., (1997), Nature Biotechnol. 15, 1359-1367).
However, in the field of plant science, only a few reports have been made on microarray analysis (Schena, M. et al., (1995), Science 270, 467-470; Ruan, Y. et al., (1998), Plant J. 15, 821-833; Abaroni. A, et al., (2000), Plant Cell 12, 647-661; and Reymond, P. et al., (2000), Plant Cell 12, 707-719).
The growth of plants is significantly affected by environmental stresses such as drought, high salinity and low temperature. Of the stresses, drought or water deficiency is the most critical factor that limits growth of plants and productions of crops. Such a drought stress causes various biochemical and physiological responses in plants.
To survive under these conditions of stress, plants acquire responsivity and adaptability to the stresses. Recently, several types of genes responsive to drought at a transcriptional level have been reported (Bohnert, H. J. et al., (1995), Plant Cell 7, 1099-1111; Ingram J., and Bartels, D. (1996), Plant Mol. Biol. 47, 377-403; Bray, E. A. (1997), Trends Plant Sci. 2, 48-54; Shinozaki, K., and Yamaguchi-Shinozaki, K. (1997), Plant Physiol. 115, 327-334; Shinozaki, K., and Yamaguchi-Shinozaki, K. (1999), “Molecular responses to drought stress. Molecular responses to cold, drought, heat and salt stress in higher plants”, edited by Shinozaki, K. and Yamaguchi-Shinozaki, K. R. G. Landes Company; and Shinozaki, K., and Yamaguchi-Shinozaki, K. (2000), Curr. Opin. Plant Biol. 3, 217-223).
On the other hand, in an attempt to improve stress resistance of plants by introducing a gene, stress-inducible genes have been used (Holmberg, J., and Bulow, L., (1998), Trends Plant Sci. 3, 61-66; and Bajaj, S. et al., (1999), Mol. Breed. 5, 493-503). Not only to further clarify the mechanism of stress resistance and stress responsivity of a higher plant at a molecular level but also to improve the stress resistance of a crop by gene manipulation, it is important to analyze the function of a stress-inducible gene.
Dehydration responsive element and C-repeat sequence (DRE/CRT) has been identified as an important cis-acting element when drought, high salt and cold stress-responsive genes are expressed in an ABA independent manner, where ABA refers to abscisic acid, a kind of plant hormone and serves as a signal transmission factor of seed dormancy and environmental stress (Yamaguchi-Shinozaki, K., and Shinozaki, K. (1994), Plant Cell 6, 251-264; Thomashow, M. F. et al., (1999), Plant Mol. Biol. 50, 571-599; and Shinozaki, K., and Yamaguchi-Shinozaki, K. (2000), Curr. Opin. Plant Biol. 3, 217-223). Furthermore, a transcriptional factor (DREB/CBF) involved in DRE/CRT responsive gene expression has been closed (Stockinger, E. J. et al., (1997), Proc. Natl. Acad. Sci. USA 94, 1035-1040; Liu, Q. et al., (1998), Plant Cell 10, 1391-1406; Shinwari, Z. K. et al., (1998), Biochem. Biophys. Res. Commun. 250, 161-170; and Gilmour, S. J. et al., (1998), Plant J. 16, 433-443). DREB1/CBF is considered to function in cold-responsive gene expression, whereas DREB2 is involved in drought-responsive gene expression. Strong resistance to freezing stress was observed in a transgenic Arabidopsis plant that overexpresses CBF1 (DREB1B) cDNA under the control of a cauliflower mosaic virus (CaMV) 35S promoter (Jaglo-Ottosen, K. R. et al., (1998), Science 280, 104-106).
The present inventors have reported that when DREB1A (CBF3) cDNA molecules are overexpressed in transgenic plants under the control of a CaMV 35S promoter or a stress-inducible rd29A promoter, strong constitutive expression of stress-inducible DREB1A target genes are induced to improve resistance to freezing, drought and salt stresses (Liu, Q. et al., (1998), Plant Cell 10, 1391-1406; and Kasuga, M. et al., (1999), Nature Biotechnol. 17, 287-291). Furthermore, the present inventors have already identified six DREB1A target genes such as rd29A/lti78/cor78, kin1, kin2/cor6.6, cor15a, rd17/cor47, and erd10 (Kasuga, M. et al., (1999), Nature Biotechnol. 17, 287-291). However, it has not yet been sufficiently elucidated how the overexpressed DREB1A cDNA improves stress resistance to freezing, drought and salt in a transgenic plant. To investigate the molecular mechanisms of drought and freezing resistance, it is important to identify and analyze as many genes controlled by DREB1A as possible.